This invention relates to compounds which are inhibitors of serine proteases and to pharmaceutical compositions thereof and their use in the treatment of the human or animal body.
The serine proteases are a group of proteolytic enzymes which have a common catalytic mechanism characterized by a particularly reactive Ser residue. Examples of serine proteases include trypsin, tryptase, chymotrypsin, elastase, thrombin, plasmin, kallikrein, Complement C1, acrosomal protease, lysosomal protease, cocoonase, xcex1-lytic protease, protease A, protease B, serine carboxypeptidase II, subtilisin, urokinase, Factor VIIa, Factor IXa, and Factor Xa. The serine proteases have been investigated extensively over a period of several decades and the therapeutic value of inhibitors of serine proteases is well understood.
Serine protease inhibitors play a central role in the regulation of a wide variety of physiological process including coagulation, fibrinolysis, fertilization, development, malignancy, neuromuscular patterning and inflammation. It is well known that these compounds inhibit a variety of circulating proteases as well as proteases that are activated or released in tissue. It is also becoming clear that serine protease inhibitors inhibit critical cellular processes, such as adhesion, migration, free radical production and apoptosis. In addition, animal experiments indicate that intravenously administered serine protease inhibitors, variants or cells expressing serine protease inhibitors, provide a protective effect against tissue damage.
Serine protease inhibitors have also been predicted to have potential beneficial uses in the treatment of disease in a wide variety of clinical areas such as oncology, neurology, hematology, pulmonary medicine, immunology, inflammation and infectious disease.
In particular serine protease inhibitors may be beneficial in the treatment of thrombotic diseases, asthma, emphysema, cirrhosis, arthritis, carcinoma, melanoma, restenois, atheroma, trauma, shock and reperfusion injury.
Thus for example an inhibitor of Factor Xa has value as a therapeutic agent as an anticoagulant, e.g. in the treatment and prevention of thrombotic disorders. The use of a Factor Xa inhibitor as an anticoagulant is desirable in view of the selectivity of its effect. Many clinically approved anticoagulants have been associated with adverse events owing to the non-specific nature of their effects on the coagulation cascade.
Also, there are well-known associations of al protease inhibitor deficiency with emphysema and cirrhosis and C1 esterase inhibitor deficiency with angioedema.
We have now found that certain novel amidine compounds are particularly effective as inhibitors of serine proteases, especially proteases with negatively charged P1 specificity pockets, and most especially the serine proteases thrombin, trypsin, urokinase and Factor Xa.
Thus viewed from one aspect we provide serine protease inhibitor compounds having an m-benzamidine group and a lipophilic group coupled to a cyclic group-attached carbon or nitrogen atom (hereinafter the alpha atom), the coupling of the m-benzamidine group to the alpha atom being by a linker group providing a two backbone atom linking chain, the backbone atoms being selected from C, N, O and S and at least one being C, optionally wherein one or both of the backbone atoms form part of a cyclic group and the coupling of the lipophilic group to the alpha atom being by a linker group capable of separating the alpha atom from the lipophilic group by a range of 2.3 to 6.5 xc3x85 in length.
In the compounds of the invention, the lipophilic group is itself optionally substituted by a hydrogen bond donor group. Where this is the case, the lipophilic group and its linker preferably are conformable to separate by from 7.5 to 15.0 xc3x85 the alpha atom and the hydrogen bond donor atom of the donor group.
Where distances from the alpha atom to the lipophilic group or to the hydrogen bond donor atom are mentioned, these relate to the distances between the centre of the alpha atom and the centre of the first atom of the lipophilic group or the centre of the hydrogen bond donor atom.
Such distances can be calculated from crystallographic data for any given compound from the bond lengths and bond angles for individual bonds along the length of the molecule between the alpha atom and the first atom of the lipophilic group or the hydrogen bond donor atom. Similarly such distances may be calculated with reasonable accuracy from the bond lengths and bond angles known to be typical of such individual bonds.
The linker between the alpha atom and the lipophilic group may itself be a lipophilic moiety, e.g. an alkylene chain. The nature of the linker may vary considerablyxe2x80x94the primary requirement is that it be conformable to place part or all of the lipophilic group at a desired distance away from the alpha atom. The lipophilic group thus desirably is able to occupy at least part of a space 2.3 to 15.0 xc3x85 from the alpha atom. The length of the linker generally corresponds to 1 to 5 backbone atoms and may be a chain, branched chain or cyclic linker (e.g. a cyclic amide or an aromatic heterocycle). In one embodiment the alpha atom forms part of a cyclic group which also forms the linker to the lipophilic group.
Thus viewed from an alternative aspect the invention provides serine protease inhibitor compounds of formula I: 
(where R1 and R2 each independently is hydrogen or hydroxyl, alkoxy, alkyl, aminoalkyl, hydroxyalkyl alkoxyalkyl, alkoxycarbonyl, acyloxymethoxycarbonyl or alkylamino optionally substituted by hydroxy, alkylamino, alkoxy, oxo, aryl or cycloalkyl;
each R3 independently is R1, R2, amino, halo, cyano, nitro, thiol, alkylthio, alkylsulphonyl, alkylsulphenyl, alkylsulphonamido, alkylaminosulphonyl, aminosulphonyl, haloalkoxy and haloalkyl;
each X independently is a C, N, O or S atom or a CO, CR1, C(R1)2 or NR1 group, at least one X being C, CO, CR1 or C(R1)2, with the proviso that if the benzamidine group is unsubstituted (i.e. no non-hydrogen R3 group is present) and the Xxe2x80x94X group is xe2x80x94CH2C(R1)2xe2x80x94 then R1 is hydrogen or is attached to the alkylene carbon atom by a heteroatom;
L is an organic linker group containing 1 to 5 backbone atoms selected from C, N, O and S, or a branched alkyl or cyclic group;
Y (the xcex1-atom) is a nitrogen atom or a CR1 group or Y and L taken together form a cyclic group;
Cy is a saturated or unsaturated, mono or poly cyclic, homo or heterocyclic group, preferably containing 5 to 10 ring atoms and optionally substituted by groups R3 or phenyl optionally substituted by R3;
Lp is a lipophilic organic group, e.g. an alkyl, heterocyclic, alkenyl, alkaryl, cycloalkyl, polycycloalkyl, cycloalkenyl, aryl, aralkyl or haloalkyl group or a combination of two or more such groups optionally substituted by one or more of oxa, oxo, aza, thio, halo, amino, hydroxy or R3 groups, preferably a group containing up to 25 carbon atoms;
D is a hydrogen bond donor group; and n is 0, 1 or 2);
or a physiologically tolerable salt thereof, e.g. a halide, phosphate or sulphate salt or a salt with ammonium or an organic amine such as ethylamine or meglumine.
In the compounds of the invention, where the alpha atom is carbon it preferably has the conformation that would result from construction from a D-xcex1-aminoacid NH2xe2x80x94CR1(Cy)xe2x80x94COOH and a m-carboxyl benzamidine. Likewise the fourth substituent R1 at an alpha carbon is preferably a methyl or hydroxymethyl group or hydrogen.
In compounds of formula (I) it is envisaged that the unsubstituted or substituted amidino group could be repalced by a substituted or unsubstituted aminomethyl group although an amidino derivative is preferred.
In the compounds of the invention, unless otherwise indicated, aryl groups preferably contain 5 to 10 ring atoms optionally including 1, 2 or 3 heteroatoms selected from O, N and S; alkyl, alkenyl or alkynyl groups or alkylene moieties preferably contain up to 6 carbons; cyclic groups preferably have ring sizes of 3 to 8 atoms; and fused multicyclic groups preferably contain 8 to 16 ring atoms.
The linker group from the benzamidine group to the alpha atom is preferably selected from xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94CONR1xe2x80x94, xe2x80x94NHxe2x80x94COxe2x80x94, xe2x80x94NHxe2x80x94CH2xe2x80x94, xe2x80x94CH2xe2x80x94NHxe2x80x94, xe2x80x94CH2Oxe2x80x94, xe2x80x94OCH2xe2x80x94, xe2x80x94COOxe2x80x94, xe2x80x94OCxe2x95x90Oxe2x80x94 and xe2x80x94CH2CH2xe2x80x94. Preferably, the X moiety nearest to the alpha atom is an NH or O atom, most preferably a NH group. The X moiety alpha to the phenyl ]ring is preferably a carbon based group such as CH2 or CO, preferably CO. Thus a particularly preferred linker Xxe2x80x94X is xe2x80x94CONHxe2x80x94.
The linker group from the alpha atom to the lipophilic group is preferably CO, CH2NH, CONR1(CH2)m, (CH2)mN(R1)CO(CH2)m, (CH2)m+2, CO(CH2)m, (CH2)mCO, (CH2)mOCxe2x95x90O, (CH2)mO or CHxe2x95x90CH(CH2)m (where each m is independently 0 or 1). The linker may be optionally branched, for example, to incorporate a polar functionality. In a preferred embodiment Y and L taken together form a cyclic group and the alpha atom is therefore a carbon atom. The cyclic group can be unsubstituted or substituted and can have a ring size of from 3 to 8 atoms. Preferably, the cyclic group is a cyclic amide, most preferably wherein the amide nitrogen of the cyclic amide group is bound to the lipophilic group.
The lipophilic group preferably comprises a cycloalkyl, azacycloalkyl, diazacycloalkyl, phenyl, naphthyl, adamantyl, decalinyl, tetrahydrodecalinyl, bicycloalkyl, mono- or diazabicycloalkyl, mono- or bicyclo heteroaromatic or a linear or branched alkyl, alkylene, alkenyl or alkenylene group all optionally substituted by one or more groups R3, or a combination of at least two such groups linked by a spiro linkage or a single or double bond or by Cxe2x95x90O, O, S, SO, SO2, CONR1, NR1xe2x80x94COxe2x80x94, NR1 linkage. For example, representative lipophilic groups include a methyl-cyclohexyl, methylcyclohexylmethyl, methylphenylmethyl, phenylethyl, benzylpiperidinyl, benzoylpiperidinyl, bispiperidinyl or phenylpiperazinyl.
Most preferably, the lipophilic group is selected from: 
wherein R3 is R1, aryl or cycloalkyl;
m represents 0 or 1;
R4 represents hydrogen, (CH2)wCOOH, (CH2)wCONH2, (CH2)wCONxcex1-AminoAcid;
w represemts an integer from 0 to 4; and
X represents CH or N.
For example specific lipophilic groups include: 
especially when R8 represents H, OMe, F, NO2, OH or Cl.
The hydrogen bond donor group which may be attached to the lipophilic group preferably has a nitrogen or oxygen atom as the donor atom and conveniently is a hydroxyl group, a primary, secondary or tertiary amine, or a primary or secondary imine group (as part of an amidine or guanidine) or a saturated or unsaturated heterocyclic group containing a ring nitrogen, preferably a group containing 5 to 7 ring atoms. Where the donor atom is a ring nitrogen, the remote portion of the heterocyclic ring may be part of the lipophilic group.
The cyclic group attached to the alpha carbon is preferably an optionally R3 substituted phenyl or naphthyl group.
The benzamidino group is preferably an unsubstituted m-benzamidino group, or a substituted m-benzamidino group bearing metabolically labile groups such as acyloxymethoxycarbonyl, alkoxycarbonyl or hydroxy.
Accordingly, preferred compounds of the invention are of formula: 
(wherein R1, R2 and R3 are as hereinbefore defined, R1 and R2 preferably being hydrogen or one representing a metabolically labile group such as alkoxycarbonyl or hydroxy, R3 preferably being hydrogen, OH or NH2 and when other than hydrogen preferably being para to the amidine group);
R5 and R6 are hydrogen or taken together with the carbon atom to which they are attached represent a carbonyl group;
Ar is an unsubstituted or substituted aryl group, preferably phenyl;
Xxe2x80x94X is xe2x80x94CONHxe2x80x94, xe2x80x94CH2CH2xe2x80x94, CH2Oxe2x80x94, xe2x80x94COOxe2x80x94, xe2x80x94CH2NHxe2x80x94, xe2x80x94OCH2xe2x80x94 or xe2x80x94NHCH2xe2x80x94;
L1 is a valence bond or an organic linker group containing 1 to 4 backbone atoms selected from C, N and O;
Lp1 is a cycloalkyl, azacycloalkyl, diazacycloalkyl, phenyl, naphthyl, adamantyl, decalinyl, tetrahydrodecalinyl, bicycloalkyl, mono- or diazabicycloalkyl, mono- or bicyclo heteroaromatic or a linear or branched alkyl, alkylene, alkenyl or alkenylene group all optionally substituted by a group R3, or a combination of at least two such groups linked by a Spiro linkage or a single or double bond or by C=O, O, S, SO, SO2, CONR1, NR1xe2x80x94COxe2x80x94, NR1 linkage. For example, representative lipophilic groups include a methylcyclohexyl, methylcyclohexylmethyl, bispiperidinyl, methylphenylmethyl, phenylethyl, benzylpiperidinyl, benzoylpiperidinyl or phenylpiperazinyl and those as hereinbefore described;
D is a hydrogen bond donor group;
and n is 0, 1 or 2).
In one embodiment, L1 comprises the backbone of an alpha amino acid, the lipophilic group being the side chain of the amino acid. The carboxyl part of the alpha amino acid may be optionally coupled via an amide bond to an amino acid or to a primary or secondary cyclic or acyclic alkyl amine or diamine or via an ester bond to primary or secondary alcohols.
In a preferred embodiment, L1 represents a valence bond and the lipophilic group is bound directly to the carbonyl alpha to the alpha atom via a nitrogen atom which forms part of the lipophilic group. Suitable lipophilic groups in this case therefore include piperidinyl, pyrrolidinyl and piperazinyl. In a preferred embodiment the piperidine or piperazinyl group is further substituted by a phenyl, benzyl, piperidine, pyridine or benzoyl group, optionally substituted on the phenyl ring by one or more R3 groups.
In a further embodiment, the lipophilic group has attached a group of the formula xe2x80x94COOR1, or xe2x80x94CON-aminoacid or ester derivative thereof.
In another embodiment the group binding the alpha carbon atom to the lipophilic group comprises a heterocyclic group. Accordingly, preferred compounds of the invention also include: 
(wherein R1, R2 and R3 are as hereinbefore defined R1 and R2 preferably being hydrogen or one representing a metabolically labile group such as alkoxycarbonyl or hydroxy, R3 preferably being hydrogen, OH or NH2 and when other than hydrogen preferably being para to the amidine group);
Ar is an unsubstituted or substituted aryl group, preferably phenyl;
Xxe2x80x94X is xe2x80x94CONHxe2x80x94, xe2x80x94CH2CH2xe2x80x94, CH2Oxe2x80x94, xe2x80x94COOxe2x80x94, xe2x80x94CH2NHxe2x80x94, xe2x80x94OCH2xe2x80x94 or xe2x80x94NHCH2xe2x80x94;
m is 0, 1 or 2;
Het is a 5 or 6-membered heterocyclic group interrupted by 1, 2 or 3 heteroatoms selected from O, N and S optionally substituted by a group R3;
Lp1 is a cycloalkyl, azacycloalkyl, diazacycloalkyl, phenyl, naphthyl, adamantyl, decalinyl, tetrahydrodecalinyl, bicycloalkyl, mono- or diazabicycloalkyl, mono- or bicyclo heteroaromatic or a linear or branched alkyl, alkylene, alkenyl or alkenylene group all optionally substituted by a group R3, or a combination of at least two such groups linked by a spiro linkage or a single or double bond or by Cxe2x95x90O, O, S, SO, SO2, CONR1, NR1xe2x80x94COxe2x80x94, NR1 linkage. For example, representative lipophilic groups include a methylcyclohexyl, methylcyclohexylmethyl, methylphenylmethyl, phenylethyl, benzylpiperidinyl, benzoylpiperidinyl, bispiperidinyl or phenylpiperazinyl;
D is a hydrogen bond donor group;
and n is 0, 1 or 2).
Where Het is a five membered ring, the two ring atoms at which it is connected are preferably separated by one ring atom. Where Het is a six-membered ring, the two ring atoms at which it is connected are preferably separated by one or two ring atoms. Representative heterocyclic groups include thiazole, oxazole, oxadiazole, triazole, thiadiazole or imidazole. Where the heterocyclic group is substituted by R3 this is preferably a COOH or COOR1 connected to the heterocycle via a valence bond or alkylene chain.
In a further embodiment, the lipophilic group has attached a group of the formula xe2x80x94COOR1 or xe2x80x94CON-aminoacid or ester derivative thereof.
The compounds of the invention may be prepared by conventional chemical synthetic routes, e.g. by amide bond formation to couple the benzamidine function to the alpha atom and to couple the lipophilic function to the alpha atom. Where the alpha atom is a carbon, the cyclic group-alpha atom combination may conveniently derive from an alpha amino acid with the benzamidine deriving from a m-amidino-benzoic acid. Amide formation from such reagents (in which the amidine function may if desired be protected during some or all of the synthesis steps) yields a compound of formula II:
Bdxe2x80x94CONHxe2x80x94CH(Cy)xe2x80x94COOHxe2x80x83xe2x80x83(II)
(where Cy is as defined above and Bd is an optionally protected m-benzamidine group).
The lipophilic group (and optionally simultaneously the hydrogen bond donor) may then conveniently be introduced by reaction of a compound of formula II (or another analogous carboxylic acid) optionally after transformation into an activated form, e.g. an acid chloride or active ester, with a lipophilic group carrying an amine, hydroxylamine, hydrazine or hydroxyl group, e.g. to produce compounds with linkages of xe2x80x94COxe2x80x94NR1xe2x80x94, xe2x80x94COxe2x80x94NR1xe2x80x94Oxe2x80x94, xe2x80x94COxe2x80x94NR1xe2x80x94NR1xe2x80x94 and xe2x80x94COxe2x80x94Oxe2x80x94 from the alpha atom (where it is a carbon) to the lipophilic group. Where Y and L taken together form a cyclic amide group the lipophilic group can be conveniently introduced by reacting the compound of formula (II) with a lipophilic group carrying a secondary amine with an active side chain. Cyclisation can be base induced via nucleophilic attack of the alpha atom on a leaving group on the active side chain. If necessary the amide linkage can be reduced using an appropriate reducing agent employing the necessary protection depending on whether concurrent reduction of the carboxylic acid moiety is also desired. Alternatively a compound of formula II or another analogous carboxylic acid may be transformed into an alcohol by reaction with isobutylchloroformate and reduction with sodium borohydride.
Such an alcohol, e.g. of formula III:
Bdxe2x80x94CONHxe2x80x94CH(Cy)CH2OHxe2x80x83xe2x80x83(III),
can be reacted to introduce the lipophilic group by reactions such as:
alkylation with an alkyl halide in the presence of a base;
reaction with diethyl azodicarboxylate/triphenylphosphine and a hydroxylated aryl compound;
by reaction with an activated carboxylic acid (e.g. an acid chloride) or with a carboxylic acid and diethylazodicarboxylate/triphenylphosphine;
by reaction with an isocyanate; and
by treatment with methanesulphonyl chloride or trifluoromethanesulphonic anhydride and reaction with an amine, or with a thiol optionally followed by oxidation, e.g. with potassium metaperiodate or hydrogen peroxide.
In this way compounds with linkages of xe2x80x94CH2xe2x80x94Oxe2x80x94, xe2x80x94CH2xe2x80x94Oxe2x80x94COxe2x80x94, xe2x80x94CH2xe2x80x94Oxe2x80x94COxe2x80x94NR1xe2x80x94, xe2x80x94CH2xe2x80x94NR1xe2x80x94, xe2x80x94CH2xe2x80x94Sxe2x80x94, xe2x80x94CH2xe2x80x94SOxe2x80x94 and xe2x80x94CH2xe2x80x94SO2xe2x80x94 between the alpha carbon and the lipophilic group may be produced.
Alternatively the alcohol can be oxidized to form a corresponding aldehyde (e.g. by oxidation with manganese dioxide or DMSO/oxalyl chloride or DMSO/SO3 or Dess-Martin reagent) which may be reacted to introduce the lipophilic group by reactions such as:
reaction with Wittig reagents or Horner-Emmons reagents, optionally followed by reduction of the resulting carbon:carbon double bond using H2/Pd-carbon;
reaction with an organometallic, eg a Grignard reagent, optionally followed by reaction on the resulting hydroxyl group, such as oxidation (eg with MnO2, DMSO/oxalyl chloride or Dess-Martin reagent), alkylation (eg with an alkyl halide in the presence of a base in a solvent such as DMF), arylation (eg with diethylazo dicarboxylate/triphenyl phosphine and a hydroxyaryl compound), ester formation (eg with an acid chloride or with a carboxylic acid and diethylazido dicarboxylate/triphenyl phosphine), or carbamate formation (eg with an isocyanate);
by reaction with an amine followed by reduction, e.g. with sodium cyanoborohydride;
by reaction with a hydrazine; or
by reaction with a carbazide.
In this way compounds with linkages of xe2x80x94CHxe2x95x90CR1xe2x80x94, xe2x80x94CH2xe2x80x94CHR1xe2x80x94, xe2x80x94CHOHxe2x80x94, xe2x80x94CHR1xe2x80x94Oxe2x80x94, xe2x80x94CHR1xe2x80x94Oxe2x80x94COxe2x80x94, xe2x80x94CHR1xe2x80x94Oxe2x80x94COxe2x80x94NR1xe2x80x94, xe2x80x94COxe2x80x94, xe2x80x94CH2xe2x80x94NR1xe2x80x94, xe2x80x94CHxe2x95x90Nxe2x80x94NR1xe2x80x94 and xe2x80x94CHxe2x95x90Nxe2x80x94NR1xe2x80x94COxe2x80x94NR1xe2x80x94 between the alpha carbon and the lipophilic group may be produced.
The transformation of alcohol to amine referred to above may be used to produce an amine reagent for lipophilic group introduction, e.g. a compound Bdxe2x80x94CONHxe2x80x94CH(Cy)xe2x80x94CH2xe2x80x94NR1H.
Such an amine reagent may be reacted to introduce the lipophilic group, e.g. by acylation with an acid halide or activated ester, by reaction with isocyanate, by reaction with an isothiocyanate, or by reaction with a sulphonyl chloride. In this way compounds with linkages of xe2x80x94CH2NR1xe2x80x94COxe2x80x94, xe2x80x94CH2xe2x80x94NR1xe2x80x94COxe2x80x94NR1xe2x80x94, xe2x80x94CH2NR1xe2x80x94CSxe2x80x94NR1xe2x80x94 and xe2x80x94CH2NR1xe2x80x94SO2xe2x80x94 between the alpha carbon and the lipophilic groups may be produced.
The transformation of acid to amide referred to above may be used to produce an amide reagent for introduction of the lipophilic group, e.g. a compound Bdxe2x80x94CONHxe2x80x94CH(Cy)xe2x80x94CON(R1)2.
Such amides may be reacted to introduce lipophilic groups, e.g. by reaction with a haloketone (e.g. phenacyl bromide). This provides a linkage: 
from alpha carbon to lipophilic group.
Analogously the amide may be transformed to a thioamide by reaction with Lawesson""s reagent and then reacted with a haloketone to form a linkage: 
The amide reagent may likewise be transformed to a nitrile reagent by dehydration, e.g. with trifluoroacetic anhydride. The nitrile reagent may be reacted with hydrazine then with acyl halide and then cyclized, (e.g. with trifluoroacetic anhydride) to produce a linkage: 
Alternatively it may be treated with hydroxylamine then reacted with acyl halide and cyclized (e.g. with trifluoroacetic anhydride) to produce a linkage: 
The hydrazide produced by reaction of a carboxylic acid reagent with hydrazine discussed above may likewise be used as a reagent for lipophilic group introduction, e.g. as a compound of formula Bdxe2x80x94CONHxe2x80x94CH(Cy)xe2x80x94COxe2x80x94NR1xe2x80x94N (R1)2.
Thus the hydrazide reagent can be reacted with an acyl halide and cyclized, e.g. with trifluoroacetic anhydride to yield a linkage: 
or reacted with an acyl halide or an isocyanate to yield linkages xe2x80x94COxe2x80x94NR1xe2x80x94NR1xe2x80x94COxe2x80x94 and xe2x80x94COxe2x80x94NR1xe2x80x94NR1xe2x80x94COxe2x80x94NR1xe2x80x94 respectively.
Alternatively the hydrazide may be transformed by reaction with Lawesson""s reagent and then reacted with an acyl halide and cyclized (e.g. with trifluoroacetic anhydride) to produce the linkage: 
An alternative route to these compounds is to carry out any of the above chemical reactions to incorporate the lipophilic group (an optional H bond donor) into a protected intermediate such as a compound of formula (IV): 
The protecting group may then be removed before coupling of the m-amidino benzoic acid (optionally protected).
A starting reagent for lipophilic group introduction where the alpha atom is nitrogen may be produced for example by reaction of a beta protected hydrazine (such protection to be chosen as to be compatible with the subsequent reagents to be employed) with phosgene, diphosgene, triphosgene or N,Nxe2x80x2 carbonyl diimidazole to give a reactive compound of the type: 
This intermediate may be used as has been described above for the carboxylic starting reagents where the alpha atom is carbon.
Removal of the protecting group by standard methods and coupling with an activated m-carboxyl-benzamidine will give compounds of the type:
xe2x80x83Bdxe2x80x94CONHxe2x80x94N(Cy)xe2x80x94Lxe2x80x94Lp(D)n
(where Bd, X, Y, Cy, L, Lp and D are as defined above).
Thus viewed from a further aspect the invention provides a process for the preparation of a compound according to the invention which process comprises coupling a lipophilic group to a compound of formula (V)
Bdxe2x80x94(X)2xe2x80x94Y(Cy)xe2x80x94Zxe2x80x83xe2x80x83(V)
(where Bd, X, Y and Cy are as defined above and Z is a reactive functional group), and optionally subsequently coupling a hydrogen bond donor group to said lipophilic group.
The compounds of the invention may be administered by any convenient route, e.g. into the gastrointestinal tract (e.g. rectally or orally), the nose, lungs, musculature or vasculature or transdermally. The compounds may be administered in any convenient administrative form, e.g. tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches etc. Such compositions may contain components conventional in pharmaceutical preparations, e.g. diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents. Preferably the compositions will be sterile and in a solution or suspension form suitable for injection or infusion. Such compositions form a further aspect of the invention. Viewed from this aspect the invention provides a pharmaceutical composition comprising a serine protease inhibitor according to the invention together with at least one pharmaceutically acceptable carrier or excipient.
Viewed from a further aspect the invention provides the use of a serine protease inhibitor according to the invention for the manufacture of a medicament for use in a method of treatment of the human or non-human animal body (e.g. a mammalian, avian or reptilian body) to combat (i.e. treat or prevent) a condition responsive to said inhibitor.
Viewed from a further aspect the invention provides a method of treatment of the human or non-human animal body (e.g. a mammalian, avian or reptilian body) to combat a condition responsive to a serine protease inhibitor (e.g. a condition such as a thrombotic disorder responsive to a factor Xa inhibitor), said method comprising administering to said body an effective amount of a serine protease inhibitor according to the invention.
The dosage of the inhibitor compound of the invention will depend upon the nature and severity of the condition being treated, the administration route and the size and species of the patient. However in general, quantities of from 0.01 to 100 xcexcmol/kg bodyweight will be administered.
All publications referred to herein are hereby incorporated by reference.
The invention will now be described further with reference to the following non-limiting Examples.
Experimental
Abbreviations used follow IUPAC-IUB nomenclature. Additional abbreviations are Hplc, high-performance liquid chromatography; DMF, dimethylformamide; DCM, dichloromethane; HOAt, 1-hydroxy-7-azabenzotriazole; HATU, [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate]; Fmoc, 9-Fluorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazole; TBTU, 2-(1H-(benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate; DIPEA, diisopropylethylamine; Boc, tertiary butyloxycarbonyl; DIPCI, diisopropylcarbodiimide; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; TEA, triethylamine; Rink linker, p-[(R,S)-xcex1-[1-(9H-Fluoren-9-yl)methoxy-formamido]-2,4-dimethoxybenzyl]phenyl acetic acid; TFA, trifluoroacetic acid; MALDI-TOF, Matrix assisted laser desorption ionisationxe2x80x94time of flight mass spectrometry; and RT, retention time. Unless otherwise indicated, amino acid derivatives, resins and coupling reagents were obtained from Novabiochem (Nottingham, UK) and other solvents and reagents from Rathburn (Walkerburn, UK) or Aldrich (Gillingham, UK) and were used without further purification.
Purification: Purification was by gradient reverse phase Hplc on a Waters Deltaprep 4000 at a flow rate of 50 ml/min. using a Deltapak C18 radial compression column (40 mmxc3x97210 mm, 10-15 mm particle size). Eluant A consisted of aqTFA (0.1%) and eluant B 90% MeCN in aqTFA(0.1%) with gradient elution (Gradient 1, 0 min. 20% B then 20% to 100% over 36 min., Gradient 2, 0 min. 5% B for 1 min. then 5% B to 20% B over 4 min., then 20% to 60% over 32 min. or Gradient 3, 0 min. 20% B then 20% to 100% over 15 min.). Fractions were analysed by analytical Hplc and MALDI-TOF before pooling those with  greater than 95% purity for lyophilisation.
Analysis: Analytical Hplc was on a Shimadzu LC6 gradient system equipped with an autosampler, a variable wavelength detector at flow rates of 0.4 ml/min. Eluents A and B as for preparative Hplc. Columns used were Techoge115 C18 (2xc3x97150 mm)(Hplc Technology), Jupiter 5 C18 column (2.1xc3x97150 mm, 5 xcexcm particle size) (Phenomenex) and Kromasil C4 (2.0xc3x97150 mm, 5 mm) HPLC Technology). Purified products were further analysed by MALDI-TOF and nmr.
All Fmoc-protected amino acids were purchased where available or prepared by known literature methods (1) with the exception of the following novel amino acid:
Preparation of (D,L)-N-Fluorenylmethyloxycarbonyl-4-phenylphenylglycine.
A solution of 4-biphenylcarboxaldehyde (4.6g 25 mmol), sodium cyanide (3.68 g 75 mmol) and ammonium carbonate (9.60 g 100 mmol) in 50% aqueous ethanol (175 ml) was heated at 50xc2x0 C. for 20 hours. The reaction mixture was cooled, concentrated under reduced pressure and acidified to pH 2.0 with concentrated hydrochloric acid (fume hood). The intermediate 4-(4-phenylphenyl)-2,5-imidazolinedione was collected by filtration, washed with dilute (0.5%) HCl and dried before using as crude in the next stage. (Filtrates were retained and treated with sodium hypochlorite solution before disposal) The intermediate 4-(4-phenylphenyl)-2,5-imidazolinedione was refluxed in 16% aqueous sodium hydroxide (100 ml, 16% w/v) for 24 h. The reaction mixture was then filtered, cooled, diluted with water (100 ml) and then shaken with ethyl acetate and separated. The aqueous solution was adjusted to pH 5.1 with concentrated hydrochloric acid and the solid obtained collected by filtration, washed with a little water and dried to give 4-phenylphenylglycine (2.36 g, 42%). 1H nmr (d6 DMSO) consistent with desired product.
To a vigorously stirred solution of 4-phenylphenylglycine (500 mg 2.2 mmol) in DCM (20 ml) was added DIPEA (614 xcexcl 4.4 mmol) and then, carefully, chlorotrimethylsilane (558 xcexcl 4.4 mmol) and the mixture refluxed for 1.5 h. The reaction mixture was cooled in an ice bath and fluorenylmethoxycarbonyl chloride (742 mg 2.2 mmol) was added in one portion. After stirring at 0xc2x0 C. for 20 min the ice bath was removed and stirring was continued for 1.5 h. The reaction mixture was concentrated under reduced pressure and the residue stirred with a mixture of diethyl ether (20 ml) and saturated sodium carbonate solution (30 ml). A yellow solid which failed to dissolve in either layer was taken up in water (20 ml) and acidified to pH 1 with dilute hydrochloric acid. The mixture was then extracted with ethyl acetate and the organic layer washed with water (2xc3x9720 ml), dried with magnesium sulphate and evaporated to dryness. Recrystallisation from ethanol/water afforded (D,L)-N-fluorenylmethyloxycarbonyl-4-phenylphenylglycine (640 mg, 65%).
1H nmr (d6 DMSO) 8.25 (1H, d, aromatics); 7.89 (2H, d, aromatics); 7.76 (2H d, aromatics); 7.67 (3H, d, aromatics); 7.57-7.25 (6H, m, aromatics); 5.23 (1H, d, xe2x80x94NH); xcx9c4.27 (3H, m, Hxcex1+CH2)
Preparation of 3-amidinobenzoic Acid TFA Salt
3 Cyanobenzoic acid (10 g, 68 mmol) was refluxed in ethanol (300 ml) on an isomantle fitted with reflux condenser and soxhlet extractor, the thimble being filled with A4 molecular sieve. Reflux was continued for 10 hours. The heating was then removed and the solution allowed to cool. The solution was then cooled in an ice bath and saturated with hydrogen chloride gas. The sealed flask was allowed to stand overnight then evaporated to dryness. To the dry product was added saturated ammonia/ethanol solution (400 ml) and the flask sealed and allowed to stand overnight. The solution was then evaporated to dryness and then treated with 2 M sodium hydroxide solution (3 eq., 102 ml ), the resulting solution was stirred for 2 hours then extracted with ethylacetate (100 ml). The aqueous layer was then acidified, with 10% aq hydrochloric acid (200 ml) in one lot and extracted with ethylacetate (100 ml). Concentrated ammonia solution was added to pH 14 and the solution cooled in the fridge overnight. The precipitate formed was filtered off and washed with water, dissolved in 10% TFA water and lyophilised to a white powder (12 g). 1H nmr (D2O) 8.40 (1H,s); 8.30 (1H, d, J=7.5 Hz); 8.00 (1H, d, J=7.5 Hz); 7.72 (1H, t)
Synthesis of Inhibitors
Method 1: Using a solid phase strategy on a Protein Technologies, Symphony Multiple Peptide Synthesiser by attachment of bis amino compounds to Peg-2-chlorotrityl chloride resin: 2-Chlorotrityl chloride resin was typically treated with greater than 2 fold excess of the di-amine in dry DCM The resin was further modified by the attachment of acids. Activation of Fmoc protected amino acid (2-5eq) was by TBTU/DIPEA, all couplings (minimum 120 min.) were carried out in DMF. Deprotection of the Fmoc group was achieved with 20% (v/v) piperidine in DMF. Other acid substituents were added as the HOBt or HOAt esters either by activation with HBTU/HATU or DIPCI with or without Boc protection of amino groups. Cleavage of the products from the resin was by treatment (30 min., ambient) with 10% (v/v) triethylsilane in TFA, filtration, evaporation and trituration with diethylether.
Synthesis using the Symphony Multiple Peptide Synthesiser.
The Symphony Multiple Peptide Synthesiser is charged with DMF, DCM, TBTU in DMF(450 mM), DIPEA in DMF (900 mM), 20% (v/v) piperidine in DMF. Resins are held in plastic reaction vessels that allow the introduction of reagents and solvents and nitrogen for agitation or air drying.
A typical synthesis cycle on the Symphony is as follows:
The reaction vessel containing the resin (0.1 mmol) is charged with the Fmoc protected amino acid (0.5 mmol) and then this is dissolved in DMF (2.5 ml), treated with TBTU (0.56 mmol, 1.25 ml) and DIPEA (1.1 mmol, 1.25 ml) and agitated with nitrogen for 2 hours (agitation times may vary). After coupling the resin is washed with DMF (6xc3x975 ml) then deprotected with 20% (v/v) piperidine in DMF (2xc3x975 ml for 1 min. each, then 1xc3x975 ml for 8 min.) the resin is then washed with DMF (6xc3x975 ml).